Project Summary Unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs) serves as the mechanistic basis for more than 25 inherited human disorders. Patients with unstable repeat expansion diseases suffer from a complex array of symptoms, including: cardiac defects, cataracts, anxiety, hyperactivity, low IQ, social deficits, respiratory defects and seizures. In some diseases, such as Fragile X Syndrome and Freidreich?s Ataxia, the downstream phenotype is mediated in large part by reduced gene expression. In all of these diseases, continuous repeat expansion is associated with disease severity. Treating trinucleotide repeat disorders is thus complex because the primary effectors of disease include both the continuous expansion of repetitive sequences as well as disrupted expression of the gene containing the repeat. Thus, an increased understanding of the molecular mechanisms governing STR instability and expansion related gene dysregulation would facilitate efforts to develop therapies to prevent and treat repeat expansion disorders. In our preliminary work, we introduce the higher order chromatin architecture as a new dimension in understanding these features in repeat expansion disorders. Our data shows that (1) the large majority of disease associated STRs are located precisely at boundaries demarcating 3D genome folding domains termed topologically associating domains (TADs) and subTADs and (2) repeat expansion in the FMR1 gene, the genetic driver for Fragile X Syndrome, results in CTCF occupancy ablation and large-scale TAD/subTAD reorganization in a manner that correlates with STR tract length, disease severity, and transcriptional disruption of FMR1. Given the increasing importance of the 3D genome, there is a critical need to extend our preliminary data to understand how the 3D genome may be perturbed by trinucleotide repeat expansion and whether this perturbation could contribute to the primary effectors of disease originating from the causal gene itself: repeat instability and dysregulated gene expression. This proposal outlines the next steps doing so. In the first aim, I will perform genome engineering experiments to determine if the domain reorganization we have observed around FMR1 contributes to decreased gene expression. In the second aim, I will create high resolution topological maps around the FXN gene, the genetic driver for Freidreich?s ataxia, the determine whether boundary disruption is present in an additional trinucleotide repeat disorder. In the third aim, I will perform additional genome editing experiments to elucidate whether domain boundary disruptions can influence repeat instability and gene expression of the FXN gene. In sum, the accomplishment of these aims would demonstrate that the 3D genome can be perturbed in repeat expansion disorders and that this perturbation can mediate repeat instability and disrupted gene expression. Ultimately, we could use these results to determine whether manipulating the 3D genome could be a potential therapeutic target for treating these diseases.